Trypsin immobilization in silica gel

Trypsin immobilization in an inorganic polymer, i.e. silica gel, was investigated. Properties of the immobilized enzyme were examined. It was found that the enzyme retained its activity, depending on the gel genesis and dehydration of the carrier. The effect of stabilizing supplements on the enzyme activity was studied. The enzyme immobilization was accompanied by an increase in its thermostability.     

Enzyme immobilization on grafted polymeric microspheres

Radiation-mediated grafting of polyacrolein onto poly(methyl methacrylate) microspheres has been shown to activate the particles for chymotrypsin (EC 3.4.21.1) immobilization. Treatment of porous polystyrene/magnetite particles with polyacrolein produced very small enzyme loading enhancement and significantly increased substrate diffusional resistance.     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Functional polymeric supports for immobilization of cholesterol oxidase

Here, we have reported the useful functional polymeric supports for possible application of enzyme immobilization. Functional polymers were prepared by free radical polymerization from different monomers (i.e., methylmetacrylate, glycidylmethacrylate, acrylamide, etc.) and N,N-methylenebis(acrylamide) (MBAAm) crosslinker. Cholesterol oxidase (ChOx) [EC.1.1.3.6] was then covalently immobilized onto these functional supports via epichlorohydrin (ECH) and carbodiimide (EDAC) as the activating agents. It was observed that, after 60th use in 5 days, the retained activities for immobilized enzymes onto poly(methyl methacrylate-co-glycidyl methacrylate) [P(MMA-co-GMA)] and poly(acrylamide-co-acrylic acid)/polyethyleneimine [P(AAm-co-AA)/PEI] supports were found as 56% and 83%, respectively.     

Covalent immobilization of the pancreatic trypsin inhibitor on a polymeric carrier

Covalent immobilization of the pancreatic trypsin inhibitor onto a polymeric carrier was accomplished by introducing a double C = C bond in the inhibitor with a subsequent copolymerization of the activized inhibitor with acrylamide and N,N'-methylenebisacrylamide. To prevent a loss of the antitryptic activity under acylation of lysine residues of the reactive centre, the inhibitor was preliminary bound to a complex with trypsin, which was destructed by acidifying the solution before copolymerization. The antitryptic activity of the immobilized inhibitor was shown to be equal to the activity of the inhibitor in the native state.     

Functionalized polymeric liposomes. Efficient immobilization of alpha chymotrypsin

A polymerizable phospholipid has been synthesized having a latent aldehyde moiety in the head group (1). Photopolymerized vesicles which have been prepared from this phosphatidylcholine have been successfully conjugated to alpha chymotrypsin. A high degree of loading was achieved with significant retention of enzymatic function.     

Protein immobilization on aminated poly(glycidyl methacrylate) nanofibers as polymeric carriers

Recently, protein carriers based on nanomaterials have been highlighted in diverse biological applications such as protein extraction, separation, and delivery due to their facile gravimetric sedimentation in the aqueous phase and abundant surface functionalities, which were used as anchoring sites for proteins. From this viewpoint, poly(glycidyl methacrylate) nanofibers (PGMA NFs) can be an excellent candidate for protein support because PGMA NFs possess the activated epoxide functional groups on the surface. In addition, cured PGMA NFs (PGMA-NH2 NFs) reveal different surface functionalities such as primary amine groups. They can be linked with carboxylated proteins. Ferritin and streptavidin were selected as models of the pristine and biolinker-mediated proteins in this experiment and immobilized onto PGMA NFs and aminated PGMA-NH2 NFs. The successful conjugations of ferritin and streptavidin were confirmed with transmission electron microscopy and fluorescein-isothiocyanate-tagged molecules. Protein immobilization using the pristine and the cured PGMA NFs could be considered as an outstanding protocol for facile protein delivery.     

An explanation for the interaction mechanism of an anionic polymeric additive on yeast flocculent cells

Summary Magna Floc LT25 is a high molecular weight anionic polymer that has been described as increasing reaction rates inside flocs of yeast cells. However, no clear indication has been given on how this anionic polymer interacts with flocculent cells. Flocculation experiments made with a strain ofSaccharomyces cerevisiae corroborate that it bridges calcium ions bound to flocculent yeast cell walls, thus enlarging the available flux area for the transport of solutes inside the flocs.     

Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres

Lipase (EC 3.1.1.3) is a versatile enzyme which has been widely used in ester-reaction industries. We have previously discovered that gigaporous polystyrene (PST) microspheres can be used as a novel immobilization carrier for lipase. In this work, a series of gigaporous microspheres with different densities of epoxy group including poly(glycidyl methacrylate) (PGMA) and poly(styrene-co-glycidyl methacrylate) [P(ST-GMA)] were evaluated as lipase immobilization carriers, which were also compared with gigaporous PST microspheres and the commercial immobilized lipase Novozym 435. Lipase immobilized in gigaporous PGMA microspheres showed the highest activity yield, reusability, and stability as well as the best affinity for the substrate. The characterizations of adsorption curves, the change of epoxy group amounts, and hydrophobic–hydrophilic properties of the microspheres were carried out to investigate the interaction between lipase molecules and carriers. It was found that covalent binding played a key role in improving the properties of lipase immobilized in gigaporous PGMA microspheres.     

胰蛋白酶的固定化及其性质研究

 以自制的脱乙酰壳多糖作载体,戊二醛为交联剂,对胰蛋白酶的固定化条件及其固定化酶的性质进行了研究。考查了交联剂的用量、pH值、以及载体与酶的比例等因素对胰蛋白酶固定化的影响。在所选择的固定化条件下,固定化酶的活性回收可达50％以上。同时研究了固定化胰蛋白酶的一些性质;最适温度60℃,最适PH8.0,Km值比可溶性酶升高,热稳定性、pH贮存稳定性以及在乙醇水溶液中的稳定性明显高于可溶性胰蛋白酶。在柱式反应器内,以2％酪蛋白为底物对,操作半衰期为40天。     

Immunoassay of low to moderately abundant anionic proteins utilizing selective immobilization to chitosan-coated plates

The quantitation of low to moderately abundant serum proteins is a common problem encountered in biochemistry. A physical property of the antigen of interest needs to be exploited in the initial binding step of an immunoassay resulting in capture (purification). We describe a two-stage immunoassay utilizing chromogenic and chemiluminescent substrates which is applied to two serum anionic proteins. In this assay, anionic serum proteins are selectively bound to positively charged chitosan-coated polystyrene plates at pH 6.1, in the presence of detergent. The assay has a detection limit of 0.1 μg/mL and a 1000-fold range.     

Preparation and characterization of UV-curable polymeric support for covalent immobilization of xylanase enzyme

The hydroxyl group of poly(ethylene glycol) monoacrylate (PEGMA) was activated by 1,1′-carbonyldiimidazole (CDI) and then a xylanase enzyme was immobilized to amine active PEGMA. UV-curable polymeric support formulation was prepared by mixing the xylanase bonded PEGMA, aliphatic polyester, 2-hydroxyethyl methacrylate (HEMA), poly(ethylene glycol) diacrylate (PEGDA) and photoinitiator. After UV irradiation, the enzymatic activity of the polymeric matrix was evaluated and compared with the corresponding free enzyme. By immobilization, the temperature resistance of the enzyme was improved and showed maximum activity at 60 °C. pH dependent activities of the free and immobilized enzymes were also investigated, and it was found that the pH of maximum activity for the free enzyme was 6.0, while for the optimal pH of the immobilized enzyme was 6.5. The immobilized enzyme retained 75% of its activity after 33 runs. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) coupled with SEM was used to explore the chemical composition. The results have confirmed the evidence of enzyme in the structure of the polymeric material.     

Enhancement of product selectivity via enzyme immobilization in sequential degradation reactions of polymeric substrates

The balance equations pertaining to the modelling of a slap-shaped bead containing immobilized enzyme uniformly distributed which catalyzes the sequential reactions of degradation of a polymeric substrate were written and analytically solved in dimensionless form. The effect of the Thiele modulus on the selectivity of consumption of each multimeric product was studied for a simple case. Whereas plain diffusional regime leads to lower selectivities than plain kinetic regime, improvements in selectivity of species A _i relative to species A_i+1 may be obtained at the expense of higher Thiele moduli within a limited range when the diffusivity of A _i is larger than that of A _i +1, or when the pseudo first order kinetic constant describing the rate of consumption of A _i is lower than that of A_i+1.List of Symbols A _i polymeric substrate containing i monomeric subunits - C _i mol·m^–3 normalized counterpart of C _i - C _i mol·m^–3 concentration of substrate A _i - C _i,0 mol·m^–3 initial concentration of substrate A _i - C _i,0 normalized counterpart of C _i,0 - D _ap,i m²·s^–1 apparent diffusivity of substrate A _i - k _i s^–1 pseudo-first order rate constant - K _m,i mol·m^–3 Michaelis-Menten constant associated with substrate A _i - L m half-thickness of the catalyst slab - N number of monomeric subunits of the largest substrate molecule - Th Thiele modulus - V _i mol·m^–3·s^–1 rate of rection of substrate A _i - V_max,i mol·m^–3·s^–1 maximum rate of reaction under saturating conditions of substrate A _i - x m longitudinal coordinate - S _i,i+1 selectivity of enzyme with respect to substrates with consecutive numbers of monomeric subunits Greek Symbols _i ratio of maximum rates of reaction - _i ratio of apparent diffusivities     

Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization.

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.     

Fabrication of polymeric ionic liquid/graphene nanocomposite for glucose oxidase immobilization and direct electrochemistry

A novel polymeric ionic liquid functionalized graphene, poly(1-vinyl-3-butylimidazolium bromide)-graphene (denoted as poly(ViBuIm(+)Br(-))-G), was synthesized. FTIR, UV-vis spectra and TEM were used to characterize the formation of as synthesized nanocomposites. Due to the modification of the polymeric ionic liquid, poly(ViBuIm(+)Br(-))-G can not only be dispersed well in aqueous solutions to form a homogeneous colloidal suspension of individual nanosheets, but also exhibit a strong positive charge. Based on self-assembly, the negatively charged glucose oxidase (GOD) was immobilized onto the poly(ViBuIm(+)Br(-))-G to form a GOD/poly(ViBuIm(+)Br(-))-G/glassy carbon (GC) electrode under mild conditions. With the advantage of both poly(ViBuIm(+)Br(-)) and graphene, poly(ViBuIm(+)Br(-))-G can provide a favorable and conductive microenvironment for the immobilized GOD and thus promote their direct electron transfer at the GC electrode. Furthermore, the GOD/poly(ViBuIm(+)Br(-))-G/GC electrode displayed an excellent sensitivity, together with a wide linear range and excellent stability for the detection of glucose. Accordingly, these unique properties of such novel nanocomposite generate a promising platform for the construction of mediator-free enzymatic biosensors.     

Gastrointestinal Transit of Soybean Trypsin Inhibitor and Fluctuations in Intraluminal Trypsin

Soybean trypsin inhibitor of Kunitz type (STI) was modified by reduced alkylation with NaBH₄ and HCHO, and examined for resistance to in vitro digestion from the viewpoint of inhibitory activity and immunoreactivity. Methylated STI was exactly alike in that respect. Hence, STI was in part replaced by its [¹⁴C]methyl-labeled specimen and their (17: 3) mixture was used as a proteinous and unabsorbable marker for digestibility experiments. When STI was immunochemically measured in intraluminal leavings in segments of the rat digestive tract, its recovery decreased progressively with the elapse of time. As a result of radioactivity measurement, however, the recovery of [¹⁴C]labeled STI proved almost quantitative all together over a period of 7 h postprandial. Concurrently, gastric emptying and intestinal transit of digesta were assessed in terms of intraluminal STI movement. After moving out from the stomach, STI rapidly passed through the upper small intestine with depression of the trypsin activity, and stayed in the lower small intestine for a few or several hours (all that while, the trypsin activity was not depressed so much). A similar pattern was observed for the intraluminal movement of a food additive ‘indigo carmine’ in another experiment. It was assumed from these observations that digesta would also have gone past the upper small bowel irrespective of ingesting either a powdered 20% casein diet or a dumpling-kneaded artificial bait.